Dioxobutanoic acid derivatives as inhibitors of influenza endonuclease

ABSTRACT

Dioxobutanoic acids substituted with piperidine or similar N-substituted saturated cycloalkyls are found to inhibit the cap-dependent endonuclease of influenza virus. These compounds are useful in the prevention or treatment of infection by influenza virus and the treatment of influenza, either as compound, pharmaceutically acceptable salts,. pharmaceutical composition ingredients, whether or not in combination with other antivirals, immunomodulators, antibiotics or vaccines. Methods of treating influenza and methods of preventing or treating infection by influenza virus are also described.

The present invention is concemed with compounds which inhibit anendonuclease encoded by influenza virus. The compounds, orpharmaceutically acceptable salts thereof, are of value in theprevention of infection by influenza virus, and the treatment ofinfection by influenza virus.

The present invention also relates to pharmaceutical compositionscontaining the compounds and to a method of use of the present compoundsand other agents for the treatment of influenza and the viral infectionby influenza virus.

BACKGROUND OF THE INVENTION

A myxovirus designated influenza is the etiological agent of the common"flu", an acute highly contagious viral disease characterized by suddenonset, fever, prostration, and progressive intimation of the respiratorymucous membrane.

The compounds of the present invention contain dioxobutanoic acidssubstituted with piperidine and similar N-substituted saturatedcycloalkyls. Applicants demonstrate that the compounds of this inventionare inhibitors of the cap-dependent endonuclease of influenza virus. Thecompounds were shown to be selective for influenza transcription bytesting in several related enzyme assays. The compounds were notinhibitory in other polymerase assays including VSV transcription, HIVreverse transcriptase, T7 phage, Hela cell RNA polymerase II and Helacell DNA polymerase α when tested at concentrations 100-500 fold abovethe IC₅₀ for influenza transcription; thereby showing specificinhibition of influenza transcription. The inhibitory activity wasspecific to cap-dependent influenza transcription and had no effect uponinfluenza transcription primed cap-independently with the dinucleotideApG. The mode of action of the inhibitors was demonstrated to beinhibition of cap-dependent endonuclease activity in an influenzacleavage assay in which the dioxobiiatanoic acids had IC_(50s) similarto those obtained in influenza trascription. Additionally, the inhibitorhad no effect upon transcription when primed with capped substrateswhich did not undergo endonucleolytic processing, further confirminginhibition of the influenza endonuclease. The specificity of influenzacleavage inhibition was demonstrated an nuclease counterscreens in whichthe compound had no effect upon RNases A, T1, U1 and HW RNase H whentested up to 100-fold above the IC₅₀ obtained in influenza cleavage.

BRIEF DESCRIPTION OF THE INVENTION

Compounds of Fomula I, as herein defined are disclosed. These compoundsare useful in the inhibition of influenza virus cap-dependentendonuclease, the prevention of infection by influenza virus, and thetreatment of infection by influenza virus, either as compounds,pharmaceutically acceptable salts, hydrates or esters, pharmaceuticalcomposition ingredients. whether or not in combination with otherantivirals, immunomodulators, antibiotics or vaccines. Methods oftreating influenza, methods of preventing infection by influenza virus,and methods of treating infection by influenza virus are also disclosed.

    ______________________________________                                        ABBREVIATIONS                                                                 ______________________________________                                        HBT (HOBT or HOBt)                                                                            Activating Agent                                                              1-hydroxybenzotriazole hydrate                                EDC             Condensing Agent                                                              1-ethyl-3-(3-dimethylamino-                                                   propyl)carbodiimide                                           ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

This invention is concemed with the compounds of Formula I, combinationsthereof, or pharmaceutically acceptable salts thereof, in the inhibitionof influenza virus transcriptase, the prevention of infection byinfluenza virus, and the treatment of infection by influenza virus.Compounds of Formula I are defined as follows: ##STR1## orpharmaceutically acceptable salt, hydrate or crystal form thereof,wherein:

X is --CH₂ --, CH₂ --CH₂ --, or a bond;

Z is --CH₂ --, CH₂ --CH₂ --, or a bond;

Y is --CH₂ --, CO,SO₂ --, or a bond;

R₁ and R₂ are independently selected from the following:

branched or unbranched C₁₋₆ alkyl-, C₁₋₆ alkyloxy-, --N--C₁₋₆ alkyl-,C₃₋₈ cycloalkyl-, phenyl, naphthyl, pyridyl, furanyl, thienyl, orquinolinyl, any of which may be substituted once or twice with C₁₋₅alkyl, C₃₋₈ cycloalkyl, phenyl, quinolinyl, pyridyl, furanyl, thienyl,C₁₋₆ -alkoxy, Br, F, or Cl.

The compounds of the present invention may have asymmetric centers andoccur as racemates, racemic mixtures and as individual diastereomers,enantiomers, or mixtures of enantiomers with all isomeric forms beingincluded in the present invention.

When any variable (e.g., R₁ or R₂, etc.) occurs more than one time inany constituent or in Formula I, its definition on each occurrence isindependent of its definition at every other occurrence. Also,combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds.

As used herein except where noted, "alkyl" is intended to include bothbranched- and straight-chain saturated aliphatic hydrocarbon groupshaving the specified number of carbon atoms (Me is methyl, Et is ethyl,Pr is propyl, Bu is butyl); "alkoxy" represents an alkyl group ofindicated number of carbon atoms attached through an oxygen bridge."Halo", as used herein, means fluoro, chloro, bromo or iodo.

The pharmaceutically-acceptable salts of the compounds of Formula I (inthe form of water- or oil-soluble or dispersible products) include theconventional non-toxic salts or the quaternary ammonium salts of thesecompounds, which are formed, e.g., from inorganic or organic acids.Examples of such acid addition salts include acetate, adipate, alginate,aspartate, benzoate, bisulfate, citrate, digluconate, dodecylsulfate,fumarate, glycerophosphate, hemisulfate, hydrochloride,2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, succinateand tartrate.

One preferred embodiment of this invention is compounds of Formula I,wherein

Y is --CH₂ --;

R₁ is

(i) phenyl, unsubstituted or substituted with halo; or

(ii) cyclohexyl;

R₂ is

(i) H or

(ii) benzyl, unsubstituted or substituted with halo;

or pharmaceutically acceptable salt, hydrate or ester thereof.

Preferred compounds of this invention include the following: ##STR2##named 4-[N-benzyl-3-(4-chlorobenzyl)piperidin-3-yl]2,4-dioxobutanoicacid hydrochloride,

or pharmaceutically acceptable salt, hydrate or ester thereof; ##STR3##named 4-[N-benzyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoicacid hydrochloride,

or pharmaceutically acceptable salt, hydrate or ester thereof; ##STR4##named 4-N-(p-chlorobenzyl )-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic acid hydrochloride,

or pharmaceutically acceptable salt, hydrate or ester thereof; ##STR5##named4-[1-cyclohexylmethyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoicacid hydrochloride,

or pharmaceutically acceptable salt, hydrate or ester thereof.

The compounds of the present invention are prepared in accordance withSchemes I-III. ##STR6##

According to Scheme I, the N-protected ester 1 is alkylated with theappropriate halide, e.g., 4-chlorobenzyl chloride, in the presence of anamide base such as LDA or LiHMDS [lithium bis(trimethylsilyl)-amide].The alkylated ester product 2 is hydrolyzed to a carboxylic acid 3.Reaction with carbonyldiimidazole affords the corresponding substitutedacyl imidazole, which reacts with the appropriate amine, e.g.,N,O-dimethylhydroxylamine, to give amide 4. Alkylation with Grignardreagent followed by deprotection of the nitrogen results in 6. Amine 6is alkylated by reaction with a slight excess of halide in base toafford 7. Reaction with an excess of reducing agent and the appropriateoxalic acid gives 8.

Scheme I is illustrated by Example 1, but is not limited to thisparticular example. ##STR7##

For 3-substituted piperidine compounds of Formula I, Scheme IIillustrates a method of synthesis. In Step C, the preferred reagent isoxalylchloride instead of carbonyldiimidazole. Otherwise, synthesis of3-substituted piperidine compounds of Formula I substantially resemblesthe synthesis of 4-substituted piperidine compounds of Formula I as setforth in Scheme I.

Scheme 2 is specifically illustrated by Example 2, but is not limited tothis particular example. ##STR8##

In Scheme III, 4-sulfonyl piperidine compounds of Formula I are readilyprepared by variations of Scheme I. The sulfonyl subsfituent can beadded first to give, for example, 17. This variation eliminates thesteps of deprotection and alkylation of the piperidinyl nitrogen (StepsE and F of Scheme I).

Scheme 3 is specifically illustrated by Example 3, but is not limited tothis particular example.

The compounds of the present invention include but are not limited tothose of the following Tables 1, 2 and 3:

                  TABLE 1                                                         ______________________________________                                         ##STR9##                                                                      ##STR10##                                                                    Example   R                   MP(°C.)                                  ______________________________________                                         2                                                                                       ##STR11##          87-90                                            3                                                                                       ##STR12##          168-170                                          4                                                                                       ##STR13##          178-180                                          5                                                                                       ##STR14##          60-62                                            6                                                                                       ##STR15##          171-173                                          7                                                                                       ##STR16##          185-186                                          8                                                                                       ##STR17##          215-218                                          9                                                                                       ##STR18##          125-131                                         10                                                                                       ##STR19##          160-162                                         11                                                                                       ##STR20##          135-140                                         12                                                                                       ##STR21##          176-178                                         13                                                                                       ##STR22##          195-197                                         14                                                                                       ##STR23##          206-208                                         15                                                                                       ##STR24##          133-136                                         16                                                                                       ##STR25##          198-200                                         17                                                                                       ##STR26##          178-180                                         18                                                                                       ##STR27##          >275                                            19                                                                                       ##STR28##          120-122                                         ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                         ##STR29##                                                                     ##STR30##                                                                    Example   R                   MP(°C.)                                  ______________________________________                                        21                                                                                       ##STR31##          160-162                                         22                                                                                       ##STR32##          55-57                                           23                                                                                       ##STR33##          70-73                                           24                                                                                       ##STR34##          168-170                                         25                                                                                       ##STR35##          148-152                                         26                                                                                       ##STR36##          190-192                                         27                                                                                       ##STR37##          189-191                                         28                                                                                       ##STR38##          184-187                                         29                                                                                       ##STR39##          117-119                                         30                                                                                       ##STR40##          74-77                                           31                                                                                       ##STR41##          154-155                                         32                                                                                       ##STR42##          120-125                                         33                                                                                       ##STR43##          70-73                                           34                                                                                       ##STR44##          209-211                                         35                                                                                       ##STR45##          130-133                                         36                                                                                       ##STR46##          108-110                                         37                                                                                       ##STR47##          167-169                                         38                                                                                       ##STR48##          212-215                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                         ##STR49##                                                                    Example   R                   MP(°C.)                                  ______________________________________                                        40                                                                                       ##STR50##          85-90                                           41                                                                                       ##STR51##          154-157                                         42                                                                                       ##STR52##          62-66                                           43                                                                                       ##STR53##          75-80                                           44                                                                                       ##STR54##          130-134                                         45                                                                                       ##STR55##           95-100                                         46                                                                                       ##STR56##          65-70                                           47                                                                                       ##STR57##          164-167                                         48                                                                                       ##STR58##          114-120                                         49                                                                                       ##STR59##          100-105                                         50                                                                                       ##STR60##          114-120                                         51                                                                                       ##STR61##          178-180                                         52                                                                                       ##STR62##          85-95                                           52                                                                                       ##STR63##          120-130                                         54                                                                                       ##STR64##          172-174                                         55                                                                                       ##STR65##           98-100                                         56                                                                                       ##STR66##          55-60                                           57                                                                                       ##STR67##          89-91                                           58                                                                                       ##STR68##          95-97                                           ______________________________________                                    

The compounds of the present invention are useful in the inhibition ofinfluenza virus cap-dependent endonuclease, and the prevention ortreatment of infection by the influenza virus.

The compounds of this invention are also useful in the preparation andexecution of screening assays for antiviral compounds. For example, thecompounds of this invention are useful for isolating enzyme mutants,which are excellent screening tools for more powerful antiviralcompounds. Furthermore, the compounds of this invention are useful inestablishing or determining the binding site of other antivirals toinfluenza virus cap-dependent endonuclease, e.g., by competitiveinhibition. Thus the compounds of this invention are commercial productsto be sold for these purposes.

For these purposes, the compounds of the present invention may beadministered orally, parenterally (including subcutaneous injections,intravenous, intramuscular, intrastemal injection or infusiontechniques), by inhalation spray, or rectally, in dosage unitformulations containing conventional non-toxicpharmaceutically-acceptable carriers, adjuvants and vehicles.

Thus, in accordance with the present invention there is further provideda method of treating and a pharmaceutical composition for treatinginfluenza virus infection and influenza. The treatment involvesadministering to a patient in need of such treatment a pharmaceuticalcomposition comprising a pharmaceutical carrier and atherapeutically-effective amount of a compound of the present invention,or a pharmaceutically-acceptable salt thereof.

These pharmaceutical compositions may be in the form oforally-administrable suspensions or tablets; nasal sprays; sterileinjectable preparations, for example, as sterile injectable aqueous oroleagenous suspensions or suppositories.

When administered orally as a suspension, these compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may contain microcrystalline cellulose for impartingbulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners/flavoring agentsknown in the art. As immediate release tablets, these compositions maycontain microcrystalline cellulose, dicalcium phosphate, starch,magnesium stearate and lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, these compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

The injectable solutions or suspensions may be formulated according toknown art, using suitable non-toxic, parenterally-acceptable diluents orsolvents, such as mannitol, 1,3-butanediol, water, Ringer's solution orisotonic sodium chloride solution, or suitable dispersing or wetting andsuspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, thesecompositions may be prepared by mixing the drug with a suitablenon-irritating excipient, such as cocoa butter, synthetic glycerideesters or polyethylene glycols, which are solid at ordinarytemperatures, but liquidify and/or dissolve in the rectal cavity torelease the drug.

Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day areuseful in the treatment or prevention of the above-indicated conditions,with oral doses two-to-five times higher. For example, infection byinfluenza virus is effectively treated by the administration of from 10to 50 milligrams of the compound per kilogram of body weight from one tothree times per day. It will be understood, however, that the specificdose level and frequency of dosage for any particular patient may bevaried and will depend upon a variety of factors including the activityof the specific compound employed, the metabolic stability and length ofaction of that compound, the age of the patient, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

The present invention is also directed to combinations of the influenzavirus inhibitory compounds with one or more agents useful in thetreatment of influenza. For example, the compounds of this invention maybe effectively administered, whether at periods of pre-exposure and/orpost-exposure, in combination with effective amounts of the influenzavirus antivirals, immunomodulators, anti-infectives, or vaccines knownto those of ordinary skill in the art.

EXAMPLE 1

4-[N-Benzyl-4-(4-Chlorobenzyl)-piperidin-4-yl]-2,4-dioxobutanoic acidHydrochloride ##STR69##

Step A Ethyl-4-(4-Chlorobenzyl)-N-Boc-isonipecotate

A solution of ethyl-N-boc-isonipecotate (51.4 g, 0.2 mole) intetrahydrofuran (1 L) at -78° C. was treated with a solution of lithiumbis (trimethylsilyl)amide in tetrahydrofuran (220 mL of a 1M solution).The solution was stirred at -60° for 15 minutes at which time4-chlorobenzyl chloride (33.8 g, 0.22 moles) was added and the reactionwarmed to room temperature over 1.5 hours. The reaction was concentratedat reduced pressure to one quarter volume and then poured into saturatedaqueous sodium bicarbonate (1 L) and extracted with ethyl acetate (2×800mL). The combined organic extracts were dried over anhydrous magnesiumsulfate, filtered, and concentrated at reduced pressure. The residue waschromatographed on silica gel with 30% ethyl acetate/hexane as eluent togive 61.3 g.

¹ H NMR CDCl₃ δ7.22 (d, J =8.0 Hz, 2H), 6.97 (d, J=8.0 Hz, 2H), 4.10 (q,J=7 Hz, 2H), 3.90 (m, 2H), 2.8 (m, 2H), 2.8 (s, 2H), 2.05 (m, 2H), 1.44(s, 9H), 1.40-1.30 (m, 2H), 1.15 (t, J =t Hz, 1H).

Step B 4-(4-Chlorobenzyl)-N-boc-isonipecotic acid

The material thus obtained in Step A was dissolved in isopropyl alcohol(150 mL) and tetrahydrofuran (150 mL) and treated with 10 N NaOH (250mL). The mixture was heated at reflux for 48 hr. The reaction was cooledto room temperature and carefully neutralized by pouting over 1 L ofcrushed ice and adding 6 N HCl until pH 3. The mixture was thenextracted with ethyl acetate (3×500 mL). The combined organic extractswere dried over anhydrous magnesium sulfate, filtered, and concentratedat reduced pressure to give 16 g of the product.

¹ H NMR CDCl₃ δ7.25 (d, J=8.5 Hz, 2H), 7.04 (d, J=8.5 Hz, 2H), 3.95 (m,2H), 2.9 (m, 2H), 2.84 (s, 2H), 2.05 (m, 2H), 1.45 (s, 9H), 1.45-1.30(m, 2H).

Step C N-boc-4-(4-Chlorobenzyl)-piperidine-4-carboxylicacid-(N,O-Dimethylmethylhydroxamide)

A solution of 4-(4-chlorobenzyl-N-boc-piperidine-4-carboxylic acid (52g, 0.131 moles) in N,N-dimethylformamide (100 mL) at room temperaturewas treated with carbonyldiimidazole (25.7 g, 0.158 mol) and warmed to60° C. for 15 minutes. N,O-dimethyl-hydroxylamine hydrochloride (30.8 g,0.316 mole) was then added and the reaction stirred at 60° C. for 15minutes. The reaction was then cooled to room temperature and aged for 1hour. After stirring for 1 hour at room temperature the reaction waspoured into saturated aqueous sodium bicarbonate and extracted withethyl acetate (3×300 mL). The combined organic extracts were dried overanhydrous magnesium sulfate, filtered, and concentrated at reducedpressure to give 58 g of the title compound.

¹ H NMR CDCl₃ δ7.20 (d, J=8.5 Hz, 2H), 6.98 (d, J=8.5 Hz, 2H), 3.93 (m,2H), 3.70 (s, 3H), 3.19 (s, 3H), 2.98 (m, 2H), 2.96 (s, 2H), 2.21 (m,2H), 1.45 (s, 9H), 1.45-1.30 (m, 2H).

Step D N-boc-4-acetyl-4-(4-Chlorobenzyl)-piperidine

A solution of N-boc-4-(4-Chlorobenzyl)-piperidine-4-carboxylicacid-(N,O-Dimethylmethylhydroxamide) (48 g, 0.120 mol) intetrahydrofuran (400 mL) was treated with a solution of methyl-magnesiumbromide (60 mL of a 3M solution in tetrahydrofuran, 0.18 mol) and heatedto reflux for thirty minutes. The reaction was then cooled to roomtemperature, poured into saturated aqueous sodium bicarbonate (1.5 L)and extracted with ethyl acetate (3×500 mL). The combined organicextracts were dried over anhydrous magnesium sulfate, filtered, andconcentrated at reduced pressure to give 25.2 g of the title compound.

¹ H NMR CDCl₃ δ7.22 (d, J=8.5 Hz, 2H), 6.96 (d, J=8.5 Hz, 2H), 3.93 (m,2H), 2.92 (m, 2H), 2.89 (s, 2H), 2.10 (s, 3H), 2.05 (m, 2H), 1.45 (s,9H), 1.50-1.30 (m, 2H).

Step E 4-Acetyl-4-(4-Chlorobenzyl)-piperidine hydrochloride

HCl gas was bubbled into a solution ofN-boc-4-acetyl-4-(4-chlorobenzyl)-piperidine (25 g, 0.07 1 mol) in ethylacetate (300 mL) for 15 minutes. The reaction was concentrated atreduced pressure and the solid collected to give 20.3 g of product.

¹ H NMR d6 DMSO δ9.1 (br s, 1H), 8.8 (br s, 1H), 7.30 (d, J=8.0 Hz, 2H),6.96 (d, J=8.0 Hz, 2H), 3.13 (m, 2H), 2.92 (s, 2H), 2.65 (m, 2H), 2.13(s, 3H), 2.05 (m, 2H), 1.70 (m, 2H).

Step F N-Benzyl-4-Acetyl-4-(4-Chlorobenzyl)-piperidine

A suspension of 4-Acetyl-4-(4-chlorobenzyl)-piperidine hydrochloride(0.600 g, 2.09 mmol) in acetonitrile (20 mL) was treated with solidsodium bicarbonate (200 mg, xs) and benzyl chloride (292 mg, 2.3 mol)and heated to reflux for 3 hours. The reaction was then cooled to roomtemperature, poured into saturated aqueous sodium bicarbonate (200 mL)and extracted with ethyl acetate (3×100 mL). The combined extracts weredried over anhydrous magnesium sulfate, filtered, and concentrated atreduced pressure. The residue was chromatographed on silica gel elutingwith 2% Methanol/chloroform to give 706 mg of the title compound.

¹ H NMR CDCl₃ δ7.25 (m, 5H), 7.22 (d, J=8.5 Hz, 2H), 6.96 (d, J=8.5 Hz,2H), 3.45 (s, 2H), 2.78 (s 2H), 2.76 (m, 2H), 2.20-2.0 (m, 4H), 2.06 (s,3H), 1.75-1.50 (m, 2H).

Step G 4-[N-Benzyl-4-(4-Chlorobenzyl)-piperidin-4-yl]-2,4-dioxo-butanoicacid Hydrochloride

A solution of N-Benzyl-4-Acetyl-4-(4-Chlorobenzyl)piperidine (600 mg,1.75 mmol) and dimethyl oxalate (310 mg, 2.63 mmol) in dimethoxyethane(20 mL) was treated with sodium hydride (105 mg of a 60% dispersion inmineral oil, 2.63 mmol) and heated to reflux for 3 hours. The reactionwas then cooled to room temperature, poured into 1N aqueous HCl (200 mL)and extracted with ethyl ether (1×50 mL). The ether extract wasdiscarded. The pH of the aqueous phase was adjusted to pH 8 andextracted with ethyl acetate (3×200 mL). The combined extracts weredried over anhydrous magnesium sulfate, filtered, and concentrated atreduced pressure. The crude ester thus obtained was dissolved in THF (5mL) and 3N HCl (20 mL) and heated to reflux for 1 hour. The reaction wasthen cooled to room temperature, and concentrated at reduced pressure.The residue was triturated with tetrahydrofuran and the solid collectedby filtration and crystallized from isopropanol to give 200 mg of thetitle compound. mp. 189°-181° C.

Elemental analysis for C₂₃ H₂₄ ClNO₄.HCl.IPA Calculated: C,60.64; H,6.56; N, 2.72 Found: C,60.61; H, 6.46; N, 2.65

EXAMPLE 24-[N-Benzyl-3-(4-Chlorobenzyl)-piperidin-3-yl]-2,4-dioxobutanoic acidHydrochloride ##STR70## Step AEthyl-3-(4-chlorobenzyl)-N-Boc-piperidin-3-carboxylate

A solution of ethyl-N-boc-nipecotate (45 g, 0.18 mole) intetrahydrofuran (200 mL) at -40° C. was treated with a solution oflithium bis (trimethylsilyl)amide in tetrahydrofuran (320 mL of a 1Msolution). The solution was stirred at -40° for 15 minutes at which time4-Chlorobenzyl chloride (34.59 g, 0.215 moles) was added and thereaction warmed to room temperature over 1.5 hours. The reaction waspoured into saturated aqueous sodium bicarbonate (1 L) and extractedwith ethyl acetate (3×500 mL). The combined organic extracts were driedover anhydrous magnesium sulfate, filtered, and concentrated at reducedpressure. The residue was chromatographed on silica gel with 30% ethylacetate/hexane as eluent to give 36.5 g.

¹ H NMR CDCl₃ δ7.22 (d, J=8.5 Hz, 2H), 7.05 (d, J=8.5 Hz, 2H), 4.1-3.8(m, 3H), 3.55 (m, 1H), 3.15 (m, 1H), 2.9 (d, J=13 Hz, 1H), 2.7 (d, J =13Hz, 1H), 2.00 (m, 1H), 1.80-1.40 (m, 3H), 1.44 (s, 9H), 1.15 (t, J=7 Hz,3H).

Step B N-boc-3-(4-chlorobenzyl)piperidin-3-carboxylic acid

A suspension of Ethyl-3-(4-chlorobenzyl)-N-Boc-piperidin-3-carboxylate(36.4 g, 0.105 mol) in isopropyl alcohol (300 mL) was treated with 10 NNaOH (300 mL). The mixture was heated at reflux for 48 hours. Thereaction was cooled to room temperature and carefully neutralized bypouting over 1 L of crushed ice and adding 6 N HCl until pH 3. Themixture was then extracted with ethyl acetate (3×500 mL). The combinedorganic extracts were dried over anhydrous magnesium sulfate, filtered,and concentrated at reduced pressure to give 31 g of the product.

¹ H NMR CDCl₃ δ10.18 (br s), 7.26 (d, J=8.5 Hz, 2H), 7.04 (d, J=8.5 Hz,2H), 3.95 (m, 1H), 3.6 (m, 1H), 3.2 (m, 2H), 2.90 (d, J=12 Hz, 1H), 2.78(d, J =12 Hz, 1H), 2.00 (m, 1H), 1.75-1.30 (m, 3H), 1.44 (s, 9H).

Step C N-boc-3-(4-Chlorobenzyl)-piperidine-3-carboxylicacid-(N,O-Dimethylmethylhydroxamide)

A solution of 3-(4-chlorobenzyl)-N-boc-nipecotic acid (23 g, 0.064moles) in methylene chloride (500 mL) at room temperature was treatedwith DMF (2 mL) and oxalyl chloride (8.5 mL), and stirred at roomtemperature for 1 hr. The reaction was then concentrated at reducedpressure and the crude acid chloride redissolved in methylene chloride(500 mL). A suspension of N,O-dimethylhydroxylamine hydrochloride (12.68g, 0.129 mole) in pyridine (50 mL) was then added and the reactionstirred at room temperature for one hour. The reaction was then pouredinto saturated aqueous sodium bicarbonate (1) and extracted with ethylacetate (3×300 mL). The combined extracts were dried over anhydrousmagnesium sulfate, filtered, and concentrated at reduced pressure togive 26.1 g of the title compound.

¹ H NMR CDCl₃ 7.15 (d, J=8.5 Hz, 2H), 7.04 (d, J=8.5 Hz, 2H), 3.95-3.6(m, 2H), 3.75 (br s, 3H), 3.2-3.0 (m, 2H), 3.0 (s, 3H), 2.98-2.6 (m,2H), 2.00-1.5 (m, 4H), 1.40 (s, 9H).

Step D N-Boc-3-acetyl-3-(4-Chlorobenzy1)-piperidine

A solution of N-boc-3-(4-Chlorobenzyl)-piperidine-3-carboxylicacid-(N,O-Dimethylmethylhydroxamide) (25.59 g, 0.064 mol) intetrahydrofuran (200 mL) was treated with a solution of methylmagnesiumbromide (64.5 mL of a 3M solution in tetrahydrofuran, 0.194 mol) andheated to reflux for thirty minutes. The reaction was then cooled toroom temperature, poured into saturated aqueous sodium bicarbonate (1 L)and extracted with ethyl acetate (3×500 mL). The combined extracts weredried over anhydrous magnesium sulfate, filtered, and concentrated atreduced pressure to give 12.2 g of the title compound.

¹ H NMR CDCl₃ δ7.25 (d,=8.5 Hz, 2H), 7.00 (d,=8.5 Hz, 2H), 4.3 (m, 1H),3.7 (m, 2H), 3.3-2.6 (m, 5H), 2.10-2.00 (m, 1H), 2.05 (s, 3H), 1.70-1.20(m, 3H), 1.45 (s, 9H).

Step E 3-Acetyl-3-(4-Chlorobenzyl)-piperidine hydrochloride

HCl gas was bubbled into a solution ofN-boc-3-acetyl-3-(4-Chlorobenzyl)-piperidine (20.46 g, 0.058 mol) inethyl acetate (300 mL) for 15 minutes. The reaction was concentrated atreduced pressure and the solid collected to give 16 g of product.

¹ H NMR d6 DMSO 15 9.1 (br s, 1H), 8.8 (br s, 1H), 7.30 (d,=8.0 Hz, 2H),6.96 (d, =8.0 Hz, 2H), 3.13 (m, 2H), 2.92 (s, 2H), 2.65 (m, 2H), 2.13(s, 3H), 2.05 (m, 2H), 1.70 (m, 2H).

Step F N-Benzyl-3-acetyl-3-(4-chlorobenzyl)-piperidine

A suspension of 3-Acetyl-3-(4-Chlorobenzyl)-piperidine hydrochloride(0.7 g, 2.43 mmol) in acetonitrile (20 mL) was treated with solid sodiumbicarbonate (200 mg, 4.8 mmol) and benzyl chloride (369 mg, 2.9 mol) andheated to reflux for 2 hours. The reaction was then cooled to roomtemperature, poured into saturated aqueous sodium bicarbonate (200 mL)and extracted with ethyl acetate (3×100 mL). The combined organicextracts were dried over anhydrous magnesium sulfate, filtered, andconcentrated at reduced pressure. The residue was chromatographed onsilica gel eluting with 2% methanol/chloroform to give 492 mg of thetitle compound.

¹ H NMR CDCl₃ δ7.40-7.25 (m, 5H), 7.22 (d,=8.5 Hz, 2H), 6.90 (d,=8.5 Hz,2H), 3.45 (m, 2H), 3.00 (m, 1H), 2.80-2.50 (m, 3H), 2.20-1.90 (m, 4H),2.00 (s, 3H), 1.60-1.50 (m, 2H), 1.30-1.10 (m, 1H).

Step G 4-[N-Benzyl-3-(4-chlorobenzyl)-piperidin-3-yl]-2,4-dioxobutanoicacid Hydrochloride

A solution of N-Benzyl-3-acetyl-3-(4-chlorobenzyl)piperidine (492 mg,1.44 mmol) and dimethyl oxalate (201 mg, 1.7 mmol) in dimethoxyethane(20 mL) was treated with sodium hydride (140 mg of a 60% dispersion inmineral oil, 3.4 mmol) and heated to reflux for 5 hours. The reactionwas then cooled to room temperature, poured into saturated sodiumbicarbonate (100 mL) and extracted with ethyl acetate (3×100 mL). Thecombined extracts were dried over anhydrous magnesium sulfate, filtered,and concentrated at reduced pressure. The crude ester thus obtained waspurified by chromatography on silica gel eluting with 10%methanol/chloroform. The material thus obtained was dissolved intetrahydrofuran (5 mL) and 3 N HCl (20 mL) and heated to reflux for 1hour. The reaction was then cooled to room temperature, and concentratedat reduced pressure. The residue was triturated with tetrahydrofuran andthe solid collected by filtration and crystallized from isopropanol togive 35 mg of the title compound. mp. 160° -165° C.

Elemental analysis for C₂₃ H₂₄ ClNO₄.HCl Calculated: C,61.34; H, 5.60;N, 3.11 Found: C,61.34; H, 5.87; N, 3.39

EXAMPLE 34-[N-Benzenesulfonyl-4-(benzyl)-piperidin-4-yl]-2,4-dioxobutanoic acid##STR71## Step A Ethyl-N-benzenesulfonyl-piperidine-4-carboxylate

A solution of ethyl isonipecotate (15.7 g, 0.1 mol) in methylenechloride (100 mL) at 0° C. was treated with pyridine (10 mL) and thenbenzenesulfonyl chloride (17.6 g, 0.1 mol). The reaction was warmed toroom temperature and stirred at room temperature for 2 hours. Thereaction was then poured into 1 N HCl (1 L) and extracted with ethylacetate (2×500 mL). The combined extracts were dried over anhydrousmagnesium sulfate, filtered, and concentrated at reduced pressure. Thesolid was recrystallized from ethyl acetate to give 17 g of product.

¹ H NMR CDCl₃ δ7.78 (d,J=6.0 Hz, 2H), 7.65-7.50 (m, 3H), 4.1 (q, J=6.8Hz, 2H), 3.62 (m, 2H), 2.50 (dr, J=3, 11.5 Hz, 2H), 2.25 (m, 1H),2.03-1.90 (m, 2H), 1.85-1.75 (m, 2H), 1.33 (t, J=6.8 Hz, 3H).

Step B Ethyl-N-Benzenesulfonyl-4-(benzyl)-piperidine-4-carboxylate

A solution of ethyl-N-benzenesulfonyl-isonipecotate (5 g, 0.016 mole) intetrahydrofuran (1 L) at -78° C. was treated with a solution of Lithiumbis (trimethylsilyl)amide in tetrahydrofuran (18.5 mL of a 1M solution).The solution was stirred at -60° for 15 minutes at which time benzylbromide (3.15 g, 0.016 moles) was added and the reaction warmed to roomtemperature over 1.5 hours. The reaction was concentrated at reducedpressure to @ one quarter volume and then poured into saturated aqueoussodium bicarbonate (1 L) and extracted with ethyl acetate (2×800 mL).The combined extracts were dried over anhydrous magnesium sulfate,filtered, and concentrated at reduced pressure. The residue waschromatographed on silica gel with 30% ethyl acetate/hexane as eluent togive 5.1 g.

¹ H NMR CDCl₁₃ δ7.7 (d,=8.5 Hz, 2H), 7.6-7.4 (m, 3H), 7.3-7.2 (m, 3H),7.1-7.0 (m, 2H), 3.98 (q, J =7 Hz, 2H), 3.65 (m, 2H), 2.78 (s, 2H), 2.35(dt, J=2.5, 12 Hz, 2H), 2.19 (d, J =12 Hz, 2H), 1.61 (dt, J=4.5, 12 Hz,2H).

Step C N-Benzenesulfonyl-4-(benzyl)-piperidine-4-carboxylic acid

The material thus obtained was dissolved in isopropyl alcoholtetrahydrofuran (150 mL) and treated with 6 N NaOH (250 mL). The mixturewas heated to reflux for 48 hours. The reaction was cooled to roomtemperature and carefully neutralized by pouting over 1 L of crushed iceand adding 6 N HCl until pH 3. The mixture was then extracted with ethylacetate (3×500 mL). The combined extracts were dried over anhydrousmagnesium sulfate, filtered, and concentrated at reduced pressure togive 4.1 g of the product.

¹ H NMR d6 DMSO δ7.70-7.60 (m, 5H), 7.30-7.15 (m, 3H), 7.06 (m, 2H),3.55 (m, 2H), 2.72 (s, 2H), 2.15 (app t, J=12 Hz, 2H), 1.98 (app d, J=12Hz, 2H), 1.50 (app dt, J=2,12 Hz, 2H).

Step D N-Benzenesulfonyl-4-(benzyl)-piperidine-4-carboxylicacid-(N,O-Dimethylmethylhydroxamide)

A solution ofN-benzenesulfonyl-4-(4-chlorobenzyl)-piperidine-4-carboxylic acid (52 g,0.131 moles) in DMF (100 mL) at room temperature was treated withcarbonyldiimidazole (25.7 g, 0.158 mol mL) and warmed to 60° C. for 15minutes. N,O-dimethylhydroxylamine hydrochloride (30.8 g, 0.316 mole)was then added and the reaction stirred at 60° C. for 15 minutes. Thereaction was then cooled to room temperature and aged for 1 hour. Afterstirring for 1 hour at room temperature the reaction was poured intosaturated aqueous sodium bicarbonate and extracted with ethyl acetate(3×300 mL). The combined extracts were dried over anhydrous magnesiumsulfate, filtered, and concentrated at reduced pressure to give 58 g ofthe title compound.

¹ H NMR CDCl₃ δ7.20 (d,=8.5 Hz, 2H), 6.98 (d,=8.5 Hz, 2H), 3.93 (m, 2H),3.70 (s, 3H), 3.19 (s, 3H), 2.98 (m, 2H), 2.96 (s, 2H), 2.21 (m, 2H),1.45 (s, 9H), 1.45-1.30 (m, 2H).

Step E N-Benzenesulfonyl-4-acetyl-4-(benzyl)-piperidine

A solution ofN-Benzenesulfonyl-4-(4-chlorobenzyl)-piperidine-4-carboxylicacid-(N,O-Dimethylmethylhydroxamide) (48 g, 0.120 mol) intetrahydrofuran (400 mL) was treated with a solution of methylmagnesiumbromide (60 mL of a 3N solution in tetra-hydrofuran, 0.18 mol) andheated to reflux for thirty minutes. The reaction was then cooled toroom temperature, poured into saturated aqueous sodium bicarbonate (1.5L) and extracted with ethyl acetate (3×500 mL). The combined extractswere dried over anhydrous magnesium sulfate, filtered, and concentratedat reduced pressure to give 25.2 g of the title compound.

¹ H NMR CDCl₃ δ7.22 (d,=8.5 Hz, 2H), 6.96 (d,=8.5 Hz, 2H), 3.93 (m, 2H),2.92 (m, 2H), 2.89 (s, 2H), 2.10 (s, 3H), 2.05 (m, 2H), 1.45 (s, 9H),1.50-1.30 (m, 2H).

Step F 4-[N-Benzenesulfonyl-4-(benzyl)-piperidin-4-yl]-2,4-dioxobutanoicacid

A solution of N-Benzyl-4-acetyl-4-(4-chlorobenzyl)piperidine (600 mg,1.75 mmol) and dimethyl oxalate (310 mg, 2.63 mmol) in dimethoxyethane(20 mL) was treated with sodium hydride (105 mg of a 60% dispersion inmineral oil, 2.63 mmol) and heated to reflux for 3 hours. The reactionwas then cooled to room temperature, poured into 1N aqueous HCI (200 mL)and extracted with ethyl ether (1×50 mL). The ether extract wasdiscarded. The pH of the aqueous phase was adjusted to pH 8 andextracted with ethyl acetate (3×200 mL). The combined extracts weredried over anhydrous magnesium sulfate, filtered, and concentrated atreduced pressure. The crude ester thus obtained was dissolved intetrahydrofuran (5 mL) and 3N NaOH (20 mL) and stirred at roomtemperature for 1 hour. The reaction was then cooled to roomtemperature, and concentrated at reduced pressure. The residue wastriturated with THF and the solid collected by filtration andcrystallized from isopropanol to give 200 mg of the title compound. mp.189°-181° C.

Elemental analysis for C₂₃ H₂₄ ClNO₄. HCl.IPA Calculated: C, 60.64; H,6.56; N, 2.72 Found: C, 60.61; H, 6.46; N, 2.65

EXAMPLE 4 SUMMARY BIOLOGICAL DATA FOR DIOXOBUTANOIC ACIDS I.IDENTIFICATION OF IN VITRO INHIBITORY ACTIVITY

The dioxobutanoic acid inhibitors were identified in an in vitro screenwhich was established for the identification of inhibitors of influenzaprimary transcription (FLUTIN) in our laboratory. The assay consisted ofdetergent-disrupted influenza virions as the enzyme source to which wasadded a capped and methylated primer, alfalfa mosaic virus (ALMV) RNAsegment 4, and ribonucleotide triphosphates, one of which wasradiolabeled to measure the incorporation into RNA by TCA precipitation.The inhibitory compounds had IC₅₀ s ranging from 0.2-29.0 μM ininfluenza transcription.

II. SPECIFIC PROTOCOLS

a. ALMV-primed flu transcription-Samples or DMSO were incubated for 60min at 31° C. in a final reaction mixture containing 20 ng/μl flu APR8virus; 2 ng/μl ALMV capped primer (880 nt); 2 ng/μl tRNA; 100 μM ATP, 50μM C and GTP, 1 μM UTP and 0.3 μM .sup.[35] S-UTP in 100 mM Tris-HCl, pH7.8/0.25% Triton-N-100/100 mM KCl/5 mM MgCl₂ /1 mM DTT. Reaction productwas quantitated by TCA precipitation on glass fiber filters, followed byliquid scintillation counting.

b. ApG-primed flu transcription-Samples or DMSO were incubated in afinal reaction mixture as in a. above, with 200 μM ApG replacing ALMVprimer.

c. VSV transcription-Samples or DMSO were incubated for 60 min at 31° C.in a final reaction mixture containing 20 ng/μl of VSV; 2 ng/μl of tRNA;100 μM ATP; 50 μM C and GTP; 1 μM UTP and 0.3 μM .sup.[35] S-UTP; in 50mM Tris-HCl, pH 8.0/100 mM NaCl/4 mM DTT/0.05% Triton-N-100/5 mM MgCl₂.Reaction product was quantitated by TCA precipitation onto glass fiberfilters, followed by liquid scintillation counting.

d. Hela RNA polymerase H-Compounds or DMSO were incubated in a run-offtranscription assay using Hela RNA polymerase II in Hela extract with 50ng/μl of pD5 template (Adeno major late promoter) in a final reactioncontaining 500 μM A,C,GTP and 0.5 μM .sup.[32] P-UTP in 15 mM Tris-HCl,pH 7.9/7.0 mM MGCl₂ /32 mM (NH₄)₂ SO₄ /0.2 mM EDTA/1.3 mM DTT for 60 minat 30° C. Following ethanol precipitation, reaction products wereelectrophoresed on 8% polyacrylamide gels containing 7M urea.

e. 13, 22, 70 nt-primed flu transcription assays-Samples or DMSO weretested in transcription as in (a) above with purified polymerase coresat a concentration of 2 ng/μl replacing APR8 virus and primed with 10 nMsynthetic ALMV capped primers (13, 22, 70 nt) replacing ALMV primer (880nt) for 40 min. Reaction was quantitated by TCA precipitation.

f. Flu Cleavage-Samples or DMSO were tested in a final reaction mixturecontaining 2.5 ng/μl of purified polymerase cores; 1 ng/μl of tRNA;0.5μ/μl of RNasin; and 5.5 nM of .sup.[32] P-radiolabeled ALMV in 50 mMTris-HCl, pH 7.8/100 mM KCl/5 mM MgCl_(2/) 1 mM DTT for 30 min. Thereaction was stopped by addition of an equal volume of 95% formamidebuffer and then electrophoresed on a 12% polyacrylamide gels containing7M urea, followed by quantitation by direct radioanalytic imaging.

g. RNase Assays-Compound or sample was tested in various RNase assays asfollows. Radiolabeled ALMV substrate as in (f), was incubated at 5.5 nMwith 0.02 units/gl of RNase T1 or 0.04 units/μl of RNase U2 in 10 mMNaCitrate, pH 5.0/0.5 mM EDTA/7M urea at 56° C. for 20 min and reactionproducts were electrophoresed on 12% polyacrylamide gels containing 7Murea as in (f). RNase A at 4 ng/μl was incubated with 5.5 mMradiolabeled ALMV substrate in 10 mM NaCitrate, pH 3.5/0.5 mM EDTA/7Murea at 30° C. for 20 min. and reaction products were electrophoresed asabove.

III. DATA FOR COMPOUND A

                  TABLE 4                                                         ______________________________________                                        BIOCHEMICAL SPECIFICITY OF COMPOUND A                                                              IC.sub.50 (μM)                                        ______________________________________                                        Polymerase                                                                    ALMV (880 nt)-primed flu transcription                                                                 1.1                                                  ApG-primed flu transcription                                                                         >1000.0                                                VSV transcription      >500.0                                                 HIV Reverse Transcriptase                                                                            >300.0                                                 T7 Phage RNA Polymerase                                                                              >100.0                                                 Hela RNA Polymerase II >500.0                                                 Hela DNA Polymerase-a  >100.0                                                 Nuclease                                                                      HIV RNase H             100.0                                                 U. sphaerogena RNase U2                                                                              >200.0                                                 A. orzae RNase T1      >200.0                                                 Bovine pancreatic RNase A                                                                            >200.0                                                 EcoRI Restriction Endonuclease                                                                       >500.0                                                 Influenza Endonuclease   1.8                                                  ______________________________________                                    

Compound A was tested in biochemical assays at concentrations in therange of 1.0-500 μM as described in materials and methods.

                  TABLE 5                                                         ______________________________________                                        INHIBITION OF INFLUENZA A AND B VIRUS                                         TRANSCRIPTION WITH COMPOUND A                                                 Virus Strain        IC.sub.50 (μM)                                         ______________________________________                                        A/PR/8/34 (H1N1)    1.10                                                      A/Japan/305/57 (H2N2)                                                                             0.25                                                      A/Port Chalmers/1/73 (H3N2)                                                                       0.50                                                      A/Hong Kong/8/68 (H3N2)                                                                           0.62                                                      B/Hong Kong/5/72    0.85                                                      ______________________________________                                    

Compound A was tested in vitro transcription with various influenzaviruses primed with cap 1 ALMV as described in material and methods.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations, or modifications, as come within thescope of the following claims and its equivalents.

What is claimed is:
 1. A compound of the Formula I: ##STR72## orpharmaceutically acceptable salt, hydrate or crystal form thereof,wherein:X is --CH₂ --, CH₂ --CH2--, or a bond; Z is --CH₂ --, CH₂ --CH₂--, or a bond; Y is --CH₂ --, CO, SO₂ --, or a bond; R₁ and R₂ areindependently selected from the following:branched or unbranched C₁₋₆alkyl-, C₁₋₆ alkyloxy-,--N--C₁₋₆ alkyl-, C₃₋₈ cycloalkyl-, phenyl,naphthyl, pyridyl, furanyl, thienyl, or quinolinyl, any of which may besubstituted once or twice with C₁₋₅ alkyl, C₃₋₈ cycloalkyl, phenyl,quinolinyl, pyridyl, furanyl, thienyl, C₁₋₆ -alkoxy, Br, F, or Cl. 2.The compound of claim 1, whereinY is --CH₂ --; R₁ is(i) phenyl,unsubstituted or substituted with halo; or (ii) cyclohexyl; R₂ is(i) Hor (ii) benzyl, unsubstituted or substituted with halo;orpharmaceutically acceptable salt, hydrate or ester thereof.
 3. Thecompound of the structure ##STR73## named4-[N-benzyl-3-(4-chlorobenzyl)piperidin-3-yl]2,4-dioxobutanoic acidhydrochloride,or pharmaceutically acceptable salt, hydrate or esterthereof;
 4. The compound of the structure ##STR74## named4-[N-benzyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic acidhydrochloride,or pharmaceutically acceptable salt, hydrate or esterthereof;
 5. The compound of the structure ##STR75## named4-N-(p-chlorobenzyl )-4-(p-chlorobenzyl)piperidin-4-yl]2,4dioxobutanoicacid hydrochloride,or pharmaceutically acceptable salt, hydrate or esterthereof;
 6. The compound of the structure ##STR76## named 4-[1-cyclohexylmethyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoicacid hydrochloride,or pharmaceutically acceptable salt, hydrate or esterthereof.